Genetic and Molecular Mechanisms Underlying Symbiotic Specificity in Legume-Rhizobium Interactions Qi Wang University of Kentucky

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3-9-2018 Genetic and Molecular Mechanisms Underlying Symbiotic Specificity in Legume-Rhizobium Interactions Qi Wang University of Kentucky

Jinge Liu University of Kentucky, [email protected]

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Repository Citation Wang, Qi; Liu, Jinge; and Zhu, Hongyan, "Genetic and Molecular Mechanisms Underlying Symbiotic Specificity in Legume- Rhizobium Interactions" (2018). Plant and Soil Sciences Faculty Publications. 105. https://uknowledge.uky.edu/pss_facpub/105

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Digital Object Identifier (DOI) https://doi.org/10.3389/fpls.2018.00313

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MINI REVIEW published: 09 March 2018 doi: 10.3389/fpls.2018.00313

Genetic and Molecular Mechanisms Underlying Symbiotic Speciﬁcity in Legume-Rhizobium Interactions

Qi Wang†, Jinge Liu† and Hongyan Zhu*

Department of Plant and Soil Sciences, University of Kentucky, Lexington, KY, United States

Legumes are able to form a symbiotic relationship with nitrogen-ﬁxing soil bacteria called rhizobia. The result of this symbiosis is to form nodules on the plant root, within which the bacteria can convert atmospheric nitrogen into ammonia that can be used by the plant. Establishment of a successful symbiosis requires the two symbiotic partners to be compatible with each other throughout the process of symbiotic development. However, incompatibility frequently occurs, such that a bacterial strain is unable to nodulate a particular host plant or forms nodules that are incapable of ﬁxing nitrogen. Genetic and

Edited by: molecular mechanisms that regulate symbiotic specificity are diverse, involving a wide Jeanne Marie Harris, range of host and bacterial genes/signals with various modes of action. In this review, University of Vermont, United States we will provide an update on our current knowledge of how the recognition specificity Reviewed by: Arijit Mukherjee, has evolved in the context of symbiosis signaling and plant immunity. University of Central Arkansas, Keywords: legume, nodulation, nitrogen fixation, rhizobial symbiosis, host specificity United States Dong Wang, University of Massachusetts Amherst, United States INTRODUCTION *Correspondence: Hongyan Zhu The legume-rhizobial symbiosis starts with a signal exchange between the host plant and its [email protected] microsymbiont (Oldroyd, 2013). Recognition of compatible bacteria by the host induces cortical †These authors have contributed cell divisions to form root nodule primordia, and simultaneously initiates an infection process to equally to this work. deliver the bacteria into the nodule cells. Infection of most legumes involves the development of plant-made infection threads that initiate in the root hair. The infection threads harboring dividing Specialty section: bacteria grow through the epidermal cell layer into the nodule cells, where the bacteria are released This article was submitted to and internalized in an endocytosis-like process. In nodule cells, individual bacteria are enclosed Plant Evolution and Development, by a membrane of plant origin, forming an organelle-like structure called the symbiosome, within a section of the journal which the bacteria further differentiate into nitrogen-fixing bacteroids (Jones et al., 2007; Oldroyd Frontiers in Plant Science et al., 2011). Received: 27 November 2017 Symbiotic nodule development involves synchronous differentiation of both nodule and Accepted: 23 February 2018 bacterial cells. Legume nodules can be grouped into two major types: indeterminate (e.g., 09 March 2018 Published: pea, clovers, and Medicago) and determinate (e.g., soybeans, common bean, and Lotus)(Nap Citation: and Bisseling, 1990; Hirsch, 1992). Indeterminate nodules originate from cell divisions in the Wang Q, Liu J and Zhu H (2018) inner cortex and possess a persistent apical meristem. Consequently, indeterminate nodules are Genetic and Molecular Mechanisms Underlying Symbiotic Specificity cylindrical in shape, with a developmental gradient from the apex to the base of the nodule, which in Legume-Rhizobium Interactions. can be divided into different nodule zones (Nap and Bisseling, 1990). In contrast, determinate Front. Plant Sci. 9:313. nodules result from cell divisions in the middle/outer cortex of the root, lack a persistent meristem, doi: 10.3389/fpls.2018.00313 and are spherical in shape. Cell divisions of a determinate nodule cease at early developmental

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stages and the mature nodule develops through cell enlargement; NodD proteins from different rhizobia are adapted to as such, the infected cells develop more or less synchronously recognizing different flavonoids secreted by different legumes, to the nitrogen-fixing stage. In both nodule types, the and this recognition specificity defines an early checkpoint of symbiotic nodule cells undergo genome endoreduplication, the symbiosis (Peck et al., 2006). Despite the absence of direct leading to polyploidization and cell enlargement. Parallel to the evidence for physical interaction between the two molecules, nodule cell development is the differentiation of the nitrogen- flavonoids have been shown to be able to stimulate the binding fixing bacteroids. Depending on the host, but independent of NodD to nod gene promoters in Sinorhizobium meliloti (Peck of the nodule type, such bacterial differentiation can be et al., 2006). It is well documented that inter-strain exchange terminal or reversible. Terminal differentiation is featured by of nodD genes can alter the response of the recipient strain to genome endoreduplication, cell elongation, increased membrane a different set of flavonoid inducers and hence the host range permeability, and loss of reproductive ability, while in reversible (Horváth et al., 1987; Perret et al., 2000). For example, the differentiation the bacteroids retain cell size and DNA content transfer of nodD1 from the broad host range symbiont Rhizobium similar to free-living bacteria (Kereszt et al., 2011; Oldroyd sp. NGR234 to the restricted host range strain Rhizobium et al., 2011; Haag et al., 2013). Compared to free-living bacteria, leguminosarum biovar trifolii ANU843 enabled the recipient the bacteroids display dramatic changes in transcriptome, cell strain to nodulate the non-legume Parasponia, because the wide- surface structure and metabolic activities so that they become host-range NodD1 protein is capable of recognizing a broader better adapted to the intracellular environment and dedicated to spectrum of flavonoid inducers (Bender et al., 1988). nitrogen fixation (Mergaert et al., 2006; Prell and Poole, 2006; The evidence for the importance of flavonoids in determining Haag et al., 2013). host range primarily comes from bacterial genetics, and the Both legumes and rhizobial bacteria are phylogenetically plant genes involved are less studied. Since legume roots secrete diverse. No single rhizobial strains can form symbiosis with a complex mixture of flavonoid compounds, it is difficult to all legumes, and vice versa. Specificity occurs at both species pinpoint which flavonoids play a more critical role, and when and genotypic levels (Broughton et al., 2000; Perret et al., 2000; and where they are produced. Recent studies in soybeans and Wang et al., 2012). This can take place at early stages of the Medicago truncatula have highlighted key flavonoids required interaction so that the same bacterial strains can infect and for rhizobial infection (reviewed in Liu and Murray, 2016). nodulate one host plant but not another (Yang et al., 2010; Wang These so called “infection flavonoids” are strong inducers of nod et al., 2012; Tang et al., 2016; Fan et al., 2017). Incompatibility genes, secreted by roots, highly accumulated at the infection also frequently happens at later stages of nodule development sites, and show increased biosynthesis in response to infection such that nitrogen-fixing efficiency differs significantly between by compatible rhizobia. Although luteolin was the first flavonoid different plant-bacteria combinations (Wang et al., 2012, 2017, identified that can induce nod gene expression across a wide 2018; Yang et al., 2017). Symbiotic specificity results from the range of rhizobial strains, it is not legume-specific, mainly changing of signals from both host and bacterial sides; as such, produced in germinating seeds, and has not been detected in various recognition mechanisms have evolved during the process root exudates or nodules. In contrast, methoxychalcone has of co-adaptation. Knowledge of the genetic and molecular basis of been shown to be one of the strong host infection signals from symbiotic specificity is important for developing tools for genetic Medicago and closely related legumes that form indeterminate manipulation of the host or bacteria in order to enhance nitrogen nodules, while genistein and daidzein are crucial signals from fixation efficiency. In this review, we will discuss our current soybeans that form determinate nodules. Part of the flavonoid understanding of the evolution of specificity in the root nodule compounds may also function as phytoalexins, acting to reinforce symbiosis. symbiosis specificity (Liu and Murray, 2016). For example, Bradyrhizobium japonicum and Mesorhizobium loti, but not the Medicago symbiont S. meliloti, are susceptible to the isoflavonoid SPECIFICITY MEDIATED BY medicarpin produced by Medicago spp. (Pankhurst and Biggs, FLAVONOIDS AND THE 1980; Breakspear et al., 2014), and the soybean symbionts FLAVONOID-NodD RECOGNITION B. japonicum and S. fredii are resistant to glyceollin when exposed to genistein and daidzein (Parniske et al., 1991). Under nitrogen-limiting conditions, legume roots secrete a cocktail of flavonoid compounds into the rhizosphere, and they serve to activate the expression of a group of bacterial SPECIFICITY MEDIATED BY nodulation (nod) genes, leading to the synthesis of the Nod factor, Nod-FACTOR PERCEPTION a lipochitooligosaccharidic signal that is essential for initiating symbiotic development in most legumes (Oldroyd et al., 2011). Nod factors produced by rhizobia are an essential signaling Induction of nod gene expression is mediated by the flavonoid- component for symbiosis development in most legumes. Nod activated NodD proteins, which are LysR-type transcription factors are lipochito-oligosaccharides, consisting of four or five regulators (Long, 1996). NodDs activate nod gene expression 1,4-linked N-acetyl-glucosamine residues that carry a fatty acyl through binding to the conserved DNA motifs (nod boxes) chain of varying length attached to the C-2 position of the non- upstream of the nod operons (Rostas et al., 1986; Fisher et al., reducing end and various species-specific chemical decorations at 1988). both the reducing and non-reducing ends (Dénarié et al., 1996).

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The common nodABC genes contribute to the synthesis of in which succinoglycan, a major EPS produced by S. meliloti, the chitin backbone, while other strain-specific nod genes act is required for the initiation and elongation of infection to modify the backbone by changing the size and saturation threads, and increased succinoglycan production enhances of the acyl chain, or adding to the terminal sugar units with nodulation capacity (Leigh et al., 1985; Reinhold et al., 1994; acetyl, methyl, carbamoyl, sulfuryl or glycosyl groups. Structural Cheng and Walker, 1998; Jones, 2012). However, the symbiotic variations in Nod factors are a key determinant of host range, role of EPS is very complicated in the Mesorhizobium-Lotus because these Nod factors have to be recognized by the host in interaction (Kelly et al., 2013). For instance, a subset of EPS order to initiate infection and nodulation (Perret et al., 2000; mutants of M. loti R7A displayed severe nodulation deficiencies D’Haeze and Holsters, 2002). on L. japonicus and L. corniculatus, whereas other mutants Nod factors are perceived by Nod-factor receptors (e.g., formed effective nodules (Kelly et al., 2013). In particular, R7A NFR1 and NFR5 in Lotus japonicus), which are LysM-domain- mutants deficient in production of an acidic octasaccharide containing receptor kinases (Limpens et al., 2003; Madsen et al., EPS were able to normally nodulate L. japonicus, while exoU 2003; Radutoiu et al., 2003). Direct binding of Nod factors mutants producing a truncated pentasaccharide EPS failed to to the extracellular LysM domains of the receptor complex invade the host. It was proposed that full-length EPS serves as a leads to activation of the downstream nodulation signaling signal to compatible hosts to modulate plant defense responses pathways (Broghammer et al., 2012). Specificity in Nod-factor and allow bacterial infection, and R7A mutants that make no binding is widely thought to be critical for recognition between EPS could avoid or suppress the plant surveillance system and the prospective symbiotic partners. This hypothesis has been therefore retain the ability to form nodules. In contrast, strains strongly supported by genetic evidence even though such binding that produce modified or truncated EPS trigger plant defense specificity has not been demonstrated. The best examples are responses resulting in block of infection (Kelly et al., 2013). from the pea-R. leguminosarum symbiosis where bacterial nod EPS production is common in rhizobial bacteria, and the gene mutants that lead to changed Nod factor composition or composition of EPS produced by different species varies widely structure exhibited genotype-specific nodulation (Firmin et al., (Skorupska et al., 2006). Several studies have suggested the 1993; Bloemberg et al., 1995). This alteration of host range involvement of the EPS structures in determining infective corresponds to allelic variations at the Sym2/Sym37/PsK1 locus, specificity (Hotter and Scott, 1991; Kannenberg et al., 1992; an orthologous region of NFR1 that contains a cluster of LysM Parniske et al., 1994; Kelly et al., 2013). Recently, an EPS receptor kinases (Zhukov et al., 2008; Li et al., 2011). In this case, receptor (EPR3) has been identified in L. japonicus, which is allelic variation coupled with gene duplication and diversification a cell surface-localized protein containing three extracellular contribute to alterations in symbiotic compatibility. LysM domains and an intracellular kinase domain (Kawaharada Nod factor recognition presumably plays a more critical role et al., 2015). EPR3 binds rhizobial EPS in a structurally specific in determining host range at species level, which has been best manner. Interestingly, Epr3 gene expression is contingent on illustrated on the bacterial side. However, natural polymorphisms Nod-factor signaling, suggesting that the bacterial entry to in Nod-factor receptors that are responsible for nodulation the host is controlled by two successive steps of receptor- specificity between different legumes have not been well studied mediated recognition of Nod factor and EPS signals (Kawaharada at the genetic level, simply because the plants cannot be interbred. et al., 2015, 2017). The receptor-ligand interaction supports Nevertheless, transferring NFR1 and NFR5 of L. japonicus into the notion that EPS recognition plays a role in regulation of M. truncatula enables nodulation of the transformants by the symbiosis specificity. However, natural variation in host-range L. japonicus symbiont Mesorhizobium loti (Radutoiu et al., specificity that results from specific recognition between host 2007). receptors and strain-specific EPS has not been demonstrated in any legume-rhizobial interactions. It is noteworthy that acidic EPS of bacterial pathogens also promote infection SPECIFICITY MEDIATED BY to cause plant disease (Newman et al., 1994; Yu et al., PERCEPTION OF RHIZOBIAL 1999; Aslam et al., 2008; Beattie, 2011). Thus, rhizobial EXOPOLYSACCHARIDES EPS might also be recognized by host immune receptors to induce defense responses that negatively regulate symbiosis In addition to Nod factors, rhizobial surface polysaccharides development. such as exopolysaccharides (EPS), lipopolysaccharides (LPS), and capsular polysaccharides (KPS) are also thought to be important for establishing symbiotic relationships (Fraysse et al., 2003; SPECIFICITY MEDIATED BY HOST Becker et al., 2005; Jones et al., 2007; Gibson et al., 2008). These INNATE IMMUNITY surface components are proposed to be able to suppress plant defense, but their active roles in promoting bacterial infection Symbiotic and pathogenic bacteria often produce similar and nodulation remain elusive and are dependent on the specific signaling molecules to facilitate their invasion of the host (Deakin interactions studied. and Broughton, 2009). These molecules include conserved Exopolysaccharides have been shown to be required for microbe-associated molecular patterns (MAMPs) and secreted rhizobial infection in multiple symbiotic interactions. This has effectors (D’Haeze and Holsters, 2004; Fauvart and Michiels, been best illustrated in the Sinorhizobium-Medicago symbiosis, 2008; Deakin and Broughton, 2009; Soto et al., 2009; Downie,

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2010; Wang et al., 2012; Okazaki et al., 2013). The host has soybeans, which depends on bacterial type III secretion system, evolved recognition mechanisms to distinguish between, and Rm41 strain lacks genes encoding such a system. It will be respond differently to, pathogens and symbionts (Bozsoki et al., interesting to know what host gene(s) control this specificity and 2017; Zipfel and Oldroyd, 2017). However, this discrimination what bacterial signals are involved. is not always successful; as a result, recognition specificity frequently occurs in both pathogenic and symbiotic interactions. In the legume-rhizobial interaction, effector- or MAMP-triggered SPECIFICITY IN NITROGEN FIXATION plant immunity mediated by host receptors also plays an important role in regulating host range of rhizobia (Yang et al., Symbiotic specificity is not confined to the early recognition 2010; Wang et al., 2012; Faruque et al., 2015; Kawaharada et al., stages; incompatible host-strain combinations can lead to 2015; Tang et al., 2016). formation of nodules that are defective in nitrogen fixation Several dominant genes have been cloned in soybeans (e.g., (Fix-). For example, a screen of a core collection of Medicago Rj2, Rfg1, and Rj4) that restrict nodulation by specific rhizobial accessions using multiple S. meliloti strains showed that ∼40% strains (Yang et al., 2010; Tang et al., 2016; Fan et al., 2017). In of the plant-strain combinations produced nodules but failed to these cases, restriction of nodulation is controlled in a similar fix nitrogen (Liu et al., 2014). The Fix- phenotype was not due manner as ‘gene-for-gene’ resistance against plant pathogens. to a lack of infection but caused by bacteroid degradation after Rj2 and Rfg1 are allelic genes that encode a typical TIR- differentiation (Yang et al., 2017; Wang et al., 2017, 2018). NBS-LRR resistance protein conferring resistance to multiple Host genetic control of nitrogen fixation specificity is B. japonicum and Sinorhizobium fredii strains (Yang et al., very complicated in the Medicago-Sinorhizobium symbiosis, 2010; Fan et al., 2017). Rj4 encodes a thaumatin-like defense- involving multiple linked loci with complex epistatic and related protein that restricts nodulation by specific strains of allelic interactions. By using the residual heterozygous lines B. elkanii (Tang et al., 2016). The function of these genes identified from a recombination inbred line population, Zhu is dependent on the bacterial type III secretion system and and colleagues were able to clone two of the underlying its secreted effectors (Krishnan et al., 2003; Okazaki et al., genes, namely NFS1 and NFS2, that regulate strain-specific 2009; Yang et al., 2010; Tsukui et al., 2013; Tsurumaru et al., nitrogen fixation concerning the S. meliloti strains Rm41 and 2015; Tang et al., 2016; Yasuda et al., 2016). These studies A145 (Wang et al., 2017, 2018; Yang et al., 2017). NFS1 and indicate an important role of effector-triggered immunity in the NFS2 both encode nodule-specific cysteine-rich (NCR) peptides regulation of nodulation specificity in soybeans. As discussed (Mergaert et al., 2003). The NFS1 and NFS2 peptides function earlier, rhizobial Nod factors and surface polysaccharides could to provoke bacterial cell death and early nodule senescence play a role in suppression of defense responses (Shaw and in an allele-specific and rhizobial strain-specific manner, and Long, 2003; D’Haeze and Holsters, 2004; Tellström et al., their function is dependent on host genetic background. NCRs 2007; Jones et al., 2008; Liang et al., 2013; Cao et al., were previously shown to be positive regulators of symbiotic 2017), but these signaling events apparently are not strong development, essential for terminal bacterial differentiation and enough to evade effector-trigged immunity in incompatible for maintenance of bacterial survival in the nodule cells (Van interactions. de Velde et al., 2010; Wang et al., 2010; Horváth et al., 2015; Many rhizobial bacteria use the type III secretion system Kim et al., 2015). The discovery of NFS1 and NFS2 revealed to deliver effectors into host cells to promote infection, and a negative role that NCRs play in regulation of symbiotic in certain situations, the delivered effector(s) are required for persistence, and showed that NCRs are host determinants of Nod-factor independent nodulation as demonstrated in the symbiotic specificity in M. truncatula and possibly also in closely soybean-B. elkanii symbiosis (Deakin and Broughton, 2009; related legumes that subject their symbiotic bacteria to terminal Okazaki et al., 2013, 2016). On the other hand, however, differentiation. recognition of the effectors by host resistance genes triggers The genomes of M. truncatula and closely related galegoid immune responses to restrict rhizobial infection. The nodulation legumes contain a large number of NCR-encoding genes that resistance genes occur frequently in natural populations, raising are expressed exclusively in the infected nodule cells (Montiel a question why host evolve and maintain such seemingly et al., 2017). These NCR genes, similar to bacterial type III unfavorable alleles. This could happen because of balancing effectors or MAMPs, can play both positive and negative roles selection, as the same alleles may also contribute to disease in symbiotic development and both roles are associated with resistance against pathogens, considering that some rhizobial the antimicrobial property of the peptides. On one hand, the effectors are homologous to those secreted by bacterial pathogens host uses this antimicrobial strategy for promoting terminal (Dai et al., 2008; Kambara et al., 2009). Alternatively, legume may bacteroid differentiation to enhance nitrogen fixation efficiency take advantage of R genes to exclude nodulation with less efficient (Oono and Denison, 2010; Oono et al., 2010; Van de Velde et al., nitrogen-fixing strains and selectively interact with strains with 2010; Wang et al., 2010). On the other hand, some rhizobial high nitrogen fixation efficiency, which is the case of the soybean strains cannot survive the antibacterial activity of certain peptide Rj4 allele. isoforms. The vulnerability of particular bacterial strains in A single dominant locus, called NS1, was also identified in response to a peptide is contingent on the genetic constitution of M. truncatula that restricts nodulation by S. meliloti strain Rm41 the bacteria as well as the genetic background of the host. It was (Liu et al., 2014). Unlike R gene-controlled host specificity in proposed that this host-strain adaptation drives the coevolution

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FIGURE 1 | Symbiosis signaling and plant immunity involved in recognition specificity in the legume-rhizobial interactions (indicated by the red stars). (A) The process of infection and nodule development. A mature indeterminate nodule contains a meristem zone (I), an infection zone (II), an interzone (IZ), a nitrogen fixing zone (III), and a senescent zone (IV). (B) The host secretes flavonoids to induce the expression of bacterial nodulation (nod) gene through the activation of NodD proteins. The enzymes encoded by the nod genes lead to the synthesis of Nod factors (NF) that are recognized by host Nod factor receptors (NFRs). Recognition specificity occurs both between Flavonoids and NodDs and between NF and NFRs. (C) In addition to NF signaling, bacteria also produce extracellular polysaccharides (EPS) and type III effectors to facilitate their infection in compatible interactions, but these molecules may also induce immune responses causing resistance to infection in incompatible interactions. (D) Certain legumes such as Medicago encode antimicrobial nodule-specific cysteine-rich (NCR) peptides to drive their bacterial partners to terminal differentiation that is required for nitrogen fixation. However, some rhizobial strains cannot survive the antibacterial activity of certain peptide isoforms, leading to formation of nodules defective in nitrogen fixation.

of both symbiotic partners, leading to the rapid amplification and that control this specificity. Work is in progress in our lab to diversification of the NCR genes in galegoid legumes (Wang et al., identify the host genes that are involved. 2017; Yang et al., 2017). Host-range specificity in the ability to fix nitrogen has also been documented in legumes (e.g., soybeans) where the bacteria CONCLUSION AND FUTURE undergo reversible differentiation. In soybeans, this type of PERSPECTIVES incompatibility was associated with the induction of phytoalexin accumulation and hypersensitive reaction in the nodule cells Specificity in the legume-rhizobial symbiosis results from a (Parniske et al., 1990). No NCR genes exist in the soybean suite of signal exchanges between the two symbiotic partners genome, implying the involvement of novel genetic mechanisms (summarized in Figure 1). Recent studies have just begun to

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reveal the underlying molecular mechanisms that regulate this be addressed. Answering these questions will certainly enrich specificity, and there are many challenging questions waiting to our knowledge of how specificity is controlled and allow us to be answered. Effector-triggered immunity has been shown to use such knowledge to develop tools for genetic improvement of be an important factor in determining host range of rhizobia symbiotic nitrogen fixation in legumes. in soybeans but the cognate effectors have not been clearly defined. In addition, what are the genes that control nodulation specificity in the Medicago-Sinorhizobium interaction where the AUTHOR CONTRIBUTIONS bacterial partner lacks the type III secretion system? Cloning and characterization of the NS1 locus in M. truncatula (Liu et al., All authors listed have made a substantial, direct and intellectual 2014) will provide novel insights into this question. We now contribution to the work, and approved it for publication. know that NCR peptides regulate nitrogen fixation specificity in Medicago and possibly in other closely related legumes, but we lack mechanistic understanding of how these peptides FUNDING work. Do the pro- and anti-symbiotic peptides interact with the same bacterial targets? How do the amino-acid substitutions This work was supported by United States Department affect the peptide structure and function? How is nitrogen of Agriculture/National Institute of Food and Agriculture, fixation specificity regulated in the NCR-lacking legumes such Agriculture and Food Research Initiative Grant 2014-67013- as soybeans where bacteria undergo reversible differentiation? 21573, Kentucky Science and Engineering Foundation Grant These are just a handful of outstanding questions that need to 2615-RDE-015, and the Kentucky Soybean Promotion Board.

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Plant conducted in the absence of any commercial or financial relationships that could Physiol. 143, 825–837. doi: 10.1104/pp.106.090985 be construed as a potential conflict of interest. Tsukui, T., Eda, S., Kaneko, T., Sato, S., Okazaki, S., Kakizaki-Chiba, K., et al. (2013). The type III secretion system of Bradyrhizobium japonicum USDA122 Copyright © 2018 Wang, Liu and Zhu. This is an open-access article distributed mediates symbiotic incompatibility with Rj2 soybean plants. Appl. Environ. under the terms of the Creative Commons Attribution License (CC BY). The use, Microbiol. 79, 1048–1051. doi: 10.1128/AEM.03297-12 distribution or reproduction in other forums is permitted, provided the original Tsurumaru, H., Hashimoto, S., Okizaki, K., Kanesaki, Y., Yoshikawa, H., author(s) and the copyright owner are credited and that the original publication and Yamakawa, T. (2015). A putative type III secretion system effector in this journal is cited, in accordance with accepted academic practice. No use, encoded by the MA20_12780 gene in Bradyrhizobium japonicum Is-34 causes distribution or reproduction is permitted which does not comply with these terms.

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